Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application.

A novel, fast and sensitive enantioselective HPLC assay with a new core-shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(-)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1-450 ng/mL (r2 ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(-)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(-)-VER established higher Cmax and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core-shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).

Electromembrane extraction of verapamil and riluzole from urine and wastewater samples using a mixture of organic solvents as a supported liquid membrane: Study on electric current variations.

In this study, the application of a mixture of organic solvents as a supported liquid membrane for improving the efficiency of the electromembrane extraction procedure was investigated. The extraction process was followed by high-performance liquid chromatography analysis of two model drugs (verapamil and riluzole). In this research, four organic solvents, including 1-heptanol, 1-octanol, 2-nitrophenyl octyl ether, and 2-ethyl hexanol, were selected as model solvents and different binary mixtures (v/v 2:1, 1:1 and 1:2) were used as the supported liquid membrane. The mixture of 2-ethyl hexanol and 1-otanol (v/v, 2:1) improved the extraction efficiency of model drugs by 1.5 to 12 times. It was found that extraction efficiency is greatly influenced by the level of electric current. In this study, for various mixtures of organic solvents, the electric current fluctuated between 50 and 2500 µA, and the highest extraction efficiencies were obtained with low and stable electric currents. Finally, the optimized extraction condition was validated and applied for the determination of model drugs in urine and wastewater samples.

Introduction of agarose gel as a green membrane in electromembrane extraction: An efficient procedure for the extraction of basic drugs with a wide range of polarities.

Developing green methods for analyte extraction is one of the most important topics in the field of sample preparation. In this study, for the first time, agarose gel was used as membrane in electromembrane extraction (EME) without using any organic solvent, for the extraction of four model basic drugs (rivastigmine (RIV), verapamil (VER), amlodipine (AML), and morphine (MOR)) with a wide polarity window (log P from 0.43 to 3.7). Different variables playing vital roles in the proposed method were evaluated and optimized. As a driving force, a 25V electrical field was applied to make the analyte migrate from sample solution with pH 7.0, through the agarose gel 3% (w/v) with 5mm thickness, into an acceptor phase (AP) with pH 2.0. The best extraction efficiency was obtained with an extraction duration of 25min. With this new methodology, MOR with high polarity (log P=0.43) was efficiently extracted without using any carrier or ion pair reagents. Limits of detection (LODs) and quantification (LOQs) were in the ranges of 1.5-1.8ngmL-1 and 5.0-6.0ngmL-1, respectively. Finally, the proposed method was successfully applied to determine concentrations of the model drugs in the wastewater sample.

Bioconcentration of two basic pharmaceuticals, verapamil and clozapine, in fish.

The present study examined the bioconcentration of 2 basic pharmaceuticals: verapamil (a calcium channel blocker) and clozapine (an antipsychotic compound) in 2 fresh water fishes, fathead minnow and channel catfish. In 4 separate bioconcentration factor (BCF) experiments (2 chemicals × 1 exposure concentration × 2 fishes), fathead minnow and channel catfish were exposed to 190 µg/L and 419 µg/L of verapamil (500 µg/L nominal) or 28.5 µg/L and 40 µg/L of clozapine (50 µg/L nominal), respectively. Bioconcentration factor experiments with fathead consisted of 28 d uptake and 14 d depuration, whereas tests conducted on catfish involved a minimized test design, with 7 d each of uptake and depuration. Fish (n = 4-5) were sampled during exposure and depuration to collect different tissues: muscle, liver, gills, kidneys, heart (verapamil tests only), brain (clozapine tests only), and blood plasma (catfish tests only). Verapamil and clozapine concentrations in various tissues of fathead and catfish were analyzed using liquid chromatography-mass spectrometry. In general, higher accumulation rates of the test compounds were observed in tissues with higher perfusion rates. Accumulation was also high in tissues relevant to pharmacological targets in mammals (i.e. heart in verapamil test and brain in the clozapine test). Tissue-specific BCFs (wet wt basis) for verapamil and clozapine ranged from 0.7 to 75 and from 31 to 1226, respectively. Tissue-specific concentration data were used to examine tissue-blood partition coefficients.

Electromembrane extraction through a virtually rotating supported liquid membrane.

Electromembrane extraction (EME) of model analytes was carried out using a virtually rotating supported liquid membrane (SLM). The virtual (nonmechanical) rotating of the SLM was achieved using a novel electrode assembly including a central electrode immersed inside the lumen of the SLM and five counter electrodes surrounding the SLM. A particular electronic circuit was designed to distribute the potential among five counter electrodes in a rotating pattern. The effect of the experimental parameters on the recovery of the extraction was investigated for verapamil (VPL), trimipramine (TRP), and clomipramine (CLP) as the model analytes and 2-ethyl hexanol as the SLM solvent. The results showed that the recovery of the extraction is a function of the angular velocity of the virtual rotation. The best results were obtained at an angular velocity of 1.83 RadS(-1) (or a rotation frequency of 0.29 Hz).The optimization of the parameters gave higher recoveries up to 50% greater than those of a conventional EME method. The rotating also allowed the extraction to be carried out at shorter time (15 min) and lower voltage (200 V) with respect to the conventional extraction. The model analytes were successfully extracted from wastewater and human urine samples with recoveries ranging from 38 to 85%. The RSD of the determinations was in the range of 12.6 to 14.8%.

Chiral separations by non-aqueous capillary electrophoresis in DMSO-based background electrolytes.

Capillary electrophoresis (CE) is a powerful technique for enantioseparations due to its high separation efficiency, high versatility, speed of analysis and low consumption of samples and reagents. Non-aqueous capillary electrophoresis (NACE) appears as a promising technique to perform enantioseparations when the drugs, chiral selectors or samples are non-water soluble. Chiral separations have been performed by NACE mainly using alcoholic solvents as BGEs, with problems of current breakdowns and changes in the BGE composition, due to their high volatility. In this work, the suitability of DMSO as BGE in NACE has been evaluated. Different experimental variables affecting the enantioresolution of three drugs have been evaluated, finally achieving complete enantioresolution of two drugs (verapamil, Rs=1.5 and pindolol, Rs=2.0) and partial resolution of the third one (fenfluramine, Rs=1.2). DMSO has been demonstrated to be a good alternative to methanolic BGEs in NACE.

Development of 2D chiral chromatography with accelerator mass spectrometry for quantification of (14)C-labeled R- and S-verapamil in plasma.

BACKGROUND: A microdose study was performed where 50 µg R/S-(14)C-verapamil was dosed intravenously to human volunteers. In order to quantify the individual R- and S-enantiomers in human plasma a 2D chiral HPLC method with subsequent analysis by accelerator mass spectrometry was verified. RESULTS: R/S-verapamil was separated on a C18 column and the isolated fraction was applied to a chiral column where the verapamil enantiomers were separated. Experimental recovery (∼73% [coefficient of variation {CV} = 16%] and 66% [CV = 21%] for R- and S-verapamil, respectively) was accounted for by the use of internal standardization from the fluorescence response of nonlabeled R- and S-verapamil. The precision of the assay ranged from 4.1 to 15.9% CV and the limit of quantitation was 1.95-4.81 pg/ml for R-verapamil and 1.76-3.34 pg/ml for S-verapamil. CONCLUSION: This method was successfully applied to the analysis of R- and S-verapamil in human plasma.

Chiral capillary electrophoretic analysis of verapamil metabolism by cytochrome P450 3A4.

Cytochrome P450 (CYP), which is one of the most important enzymes in human liver, is responsible for a large portion of the first-pass metabolism of drugs. Many studies have focused on the determination of CYP activity by substrate assays. Most of them used liquid chromatography (LC) as analytical technique, while only a few studies used capillary electrophoresis (CE) for the separation and quantitation of reaction components. In this study, the feasibility of using CE in an in vitro metabolism study with CYP was tested. Verapamil was chosen as the substrate for CYP 3A4 isozyme (Supersome). A chiral capillary electrophoretic method was developed and validated for the simultaneous determination of R,S-verapamil (VER) and their major metabolites, R,S-norverapamil (NOR). A method for CYP 3A4 activity assay was proposed with VER as a probe. At the same time, the enantioselective metabolism of VER was studied. Michaelis-Menten constants of R- and S-VER were determined. S-VER was metabolised faster and more extensively than R-VER, with K(m)=167+/-23 microM, V(max)=3,418+/-234 pmol/min/mg for S-VER, and K(m)=168+/-35 microM, V(max)=2,502+/-275 pmol/min/mg for R-VER.

High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase.

The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Brönsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.

Thin-layer chromatography separation of enantiomers of verapamil using macrocyclic antibiotic as a chiral selector.

Silica gel thin-layer chromatography plates impregnated with macrocyclic antibiotic, vancomycin, as chiral selector were prepared and used for the resolution of (+/-)-verapamil. A mobile phase system of acetonitrile-methanol-water (15:2.5:2.5, v/v) was worked out systematically. The effects of chiral selector, temperature and pH on resolution were also studied. The spots were detected with iodine vapors and the detection limit was found to be 0.074 microg of each enantiomers.

An improved method for the preparation of [11C]verapamil.

This paper describes an improved preparation of [11C]verapamil by reaction of [11C]methyl triflate with desmethylverapamil. The optimal reaction temperature, amount of precursor and reaction time were assessed. With this method [11C]verapamil can be prepared with a reproducible radiochemical yield of 66 +/- 4% (EOB, based on [11C]methyltriflate). Total synthesis time was 60 min. Radiochemical purity was >99% and specific activities varied between 5 and 30TBq/mmol.

Validation of a capillary electrophoresis assay for assessing the metabolic stability of verapamil in human liver microsomes.

A simple CE assay for the rapid determination of the in vitro metabolic stability of verapamil in human liver microsomes has been developed and validated. Verapamil was used as the prototype drug since it is extensively metabolized in human liver microsomes. The assay showed good intra- (CV < or = 10%) and interday (CV < or = 8%) reproducibility. The recovery of verapamil after incubation at 37 degrees C for 60 min with human liver microsomes was low (15 +/- 1%) and two metabolites were detected. The method is currently in use for assessing the metabolic stability of new drug candidates at an early stage of lead optimization at Cardiome Pharma Corp. (Vancouver, BC, Canada).

Chiral separation of verapamil and some of its metabolites by HPLC and CE.

HPLC and CE assays were developed for chiral separations of verapamil and its metabolites in serum samples. Three chiral HPLC columns (Chiralcel OJ, Chiralpak AD and Chiralcel OD-R) were tested in normal and reverse-phase modes. All HPLC analyses were performed with fluorescence detection at 276 and 310 nm. CE was realized using CM-beta-CD as a chiral selector for the enantiomeric analysis. The results of HPLC and CE studies were compared and the possibilities for the applications in therapeutic drug monitoring were discussed.

Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals.

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed-phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest and most reproducible recoveries were obtained using a 30 mg Oasis-HLB sorbent and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused-silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.

Enantiomer separation by strong anion-exchange capillary electrochromatography with dynamically modified sulfated beta-cyclodextrin.

A novel mode of capillary electrochromatography (CEC) based on a dynamically modified stationary phase was presented for chiral separation. The capillary column was packed with strong anion-exchange (SAX) stationary phase packing; the sulfated beta-cyclodextrin (S-CD), which was added to the mobile phase, was dynamically adsorbed to the packing surface. Separation of enantiomers was achieved by their different abilities to form an inclusion complex with the adsorbed S-CD. The enantiomers of tryptophan, praziquantel, atropine, metoprolol, and verapamil were successfully separated in this system with a column efficiency of 36000-412000 plates/m. The resolution value obtained for atropine was as high as 11.23. The superiority of CEC with a dynamically modified stationary phase over that with a physically adsorbed stationary phase was demonstrated. The influence of ionic strength, S-CD concentration, and methanol content on separation was also studied.

[Enantioseparation of twelve pharmaceutical racemates with high performance capillary electrophoresis using L-leucine as chiral selector].

A rapid enantiomeric separation method using L-leucine as chiral selector was established. Capillary zone electrophoresis (CZE) has been used for the enantiomeric separation of twelve pharmaceutical racemates with bare fused silica capillary and employing L-leucine as chiral selector. The enantiomeric resolution was influenced by L-leucine concentration and pH of background electrolyte (BGE). The effects of the BGE types and concentrations on the enantiomeric separation were also investigated. The results showed that in the solution containing 50 mmol/L borax and 70 mmol/L L-leucine (pH 9.0), all the twelve drugs were on baseline separated in less than 11 minutes.

Study of enantioselective interactions between chiral drugs and serum albumin by capillary electrophoresis.

The separation of the enantiomers of three basic drugs, i.e., ofloxacin, propranolol and verapamil, was achieved by affinity capillary electrophoresis (ACE), with human serum albumin (HSA) and bovine serum albumin (BSA) as chiral selectors in phosphate buffer at pH 7.4. Ofloxacin was only separated in the presence of BSA, and verapamil only with HSA, while propranolol was separated with either HSA or BSA. The effects of protein concentration and column wall adsorption on the degree of separation were investigated. Two displacers, ketoprofen and warfarin, respectively, when added to the protein containing buffer, both showed significant effects on the separation behavior. From these data it was argued that verapamil may bind to HSA at both locations known, the warfarin binding site (I) and the ketoprofen binding site (II). While with BSA, binding of ofloxacin may also occur at site I, the preferential binding site for propanolol remains controversial. A drug-drug interaction between propranolol and ketoprofen due to opposite charges was concluded from the increase in migration time in BSA solution. The unbound concentration of verapamil enantiomers in solution in the presence of HSA, as estimated from CD-modified capillary zone electrophoresis, was triggered not only by the HSA concentration but also by the coadditive concentration.

Enantiometric separation of verapamil and norverapamil using Chiral-AGP as the stationary phase.

Simultaneous enantiomeric separation of verapamil and its main metabolite norverapamil was achieved using Chiral-AGP as the stationary phase. The optimized chromatographic system was obtained using statistical experimental design with partial least squares as regression method. The three variables studied were buffer pH, content of acetonitrile and column temperature. A high buffer pH favors enantioselectivity as well as the selectivity between (S)-verapamil and (R)-norverapamil. The concentration of the organic modifier in the mobile phase was a compromise as a high content of acetonitrile decreased enantioselectivity but increased the selectivity mentioned above. Increased column temperature increased the separation between (S)-verapamil and (R)-norverapamil with only a slight decrease in enantioresolution.

[A novel chiral selector in capillary electrophoresis--beta-cyclodextrin polymer].

Three chiral selectors CM-beta-CD, EP-beta-CD, beta-CD were studied for the enatio-separation of three drugs under optimum conditions respectively. The results demonstrate that the resolving power for the drugs is as follows CM-beta-CD > EP-beta-CD > beta-CD, with the exception of lobeline. This is due to the--CH2COOCH3 group of CM-beta-CD, which will change the combination and improve the recognition on guest molecules. Although EP-beta-CD is inferior to CM-beta-CD for the separation of chlorpheniramine and verapamil, it has excellent recognition on lobeline and it has not been reported previously. In most cases EP-beta-CD is superior to beta-CD. The explanations are: (1) EP-beta-CD has good solubility in water, which enables high concentrations to be used and consequently achieves excellant separation of racemic compounds, (2) the polymerization of beta-CD changes the properties of CD units and the process produces a more rigid and different conformation from CD, (3) we must attribute much merits to the cooperation or synergism of two, three or even more CD moieties of two polymers for inclusion. Complexation with analytes possesses more than one guest part in their structure.

Column-switching techniques in the biomedical analysis of stereoisomeric drugs: why, how and when.

The application of stereoselective chromatographic techniques to bioanalytical problems has become a routine procedure. However, this approach is not always straightforward; particularly when the separation involves chromatographic chiral stationary phases. Matrix interferences and more importantly, overlapping metabolite peaks often make direct analysis impractical. One strategy to overcome these problems is to combine two or more columns with different selectivities to produce a multi-dimensional chromatographic system. This review addresses the use of coupled column chromatography in HPLC systems including different coupling methods and the application of the resulting arrangements to bioanalytical analyses.